The goal of our research is to understand better the genetic and molecular basis of bacterial invasion. In particular, we propose to focus on the identification and characterization of Salmonella typhimurium genetic sequences that are expressed within infected host cells and within infected animals. We have developed a new method termed differential fluorescence induction, which utilizes green fluorescent protein and a fluorescent activated sorter (FACS) to identify bacterial genes that are expressed exclusively within animal cells or during infection. We will focus on macrophage-inducible promoter sequences (mig) and animal-induced genes (aig) isolated from S. Typhimurium SL1344 by sorting cells (by FACS) from the tissue of infected animals or from infected cultured cells. Four mig promoter sequences we have identified thus far represent distinct general categories of genes associated with intracellular growth and persistence. We propose several strategies to determine the potential function of these genetic sequences using the methods of multi-parametric FACS analysis and confocal microscopy to analyze intracellular trafficking. Finally, we propose to isolate variants of the mig and aig genes to follow the fate of wildtype and avirulent mutant organisms in the tissues of infected animals. Thus, our aim is to systematically examine gene expression within macrophages and infected animals using the mutant strains we create, the GFT genetic fusions we construct, and the resolving power of FACS cell sorting to discover genes essential for the pathogenesis of infection. We believe that these principles can be broadly applied to the study of a number of infectious diseases.